The development of new drug formulations for physiologically active peptides and proteins is focused on maintaining biological activity but even these are limited by the inherently short half-life or instability of the peptides and proteins in the body. This is especially true for small peptides and proteins with a hydrodynamic diameter of less than about 5 nm. There has long been a desire to alleviate such short half-life or instability of peptides and proteins in the body either by the use of infusion devices that constantly deliver rapidly degrading peptide or protein drugs or by providing an eroding depot of the drug under the skin. Development of excipients that can extend the half-life and/or provide stability of the peptides and proteins in the body and blood is a new area of research.
Liposomes that entrap unstable or short half-life drugs rely on the degradation of the liposome structure before the drug can be released. Polylactic-co-glycolic acid particles are another entrapment technology that relies on enzyme degradation of the polymer to release the drug. Semi-random grafting of two or more polymers that results in a co-polymer that binds rather than entraps the drug has been done (U.S. application Ser. Nos. 11/613,183, 11/971,482, and Castillo et al. Pharm. Res. 2012 Vol 29(1) p 306-318). Such co-polymers provide blood stability, extension of half-life, and prolonged elevated blood level of administered peptides and proteins (U.S. application Ser. Nos. 11/613,183, 11/971,482, and Castillo et al. Pharm. Res. 2012 Vol 29(1) p 306-318).
However, the ability to improve upon the manufacturing process and the potency of the grafted co-polymer product (the capacity to bind peptide on a per weight basis) is limited by the absence of technology that can determine the exact organization and periodicity, if any, in which the two polymers are grafted to the other polymer and relative to each other. It appears that there is a process-induced determinant of the organization of the components of the co-polymer molecule that then defines the final co-polymer product composition and properties. Because the compositional organization of the final product cannot be evaluated using the existing technology, the product can only be defined by the processes used to manufacture said product along with the product-associated properties that distinguishes said product from other products that are made using similar but not identical processes. Identification of such a process that makes a superior product is not obvious because of the lack of analytical technology that elucidates the atomic organization of the product and relying on the experimentation of various processes and evaluating the potency of the final product can take many years of detailed trial and error experimentation. The differences in composition of the final products can only be determined by their potency which can be defined by the process by which they are made. This is because the polymers are large, the co-polymerization reaction is random, and, as the reaction proceeds, the conformation of the polymers being grafted can change resulting in a non-random distribution that is determined by conformation at any given moment of the reaction timeline. The change in conformation is especially true with polylysine, which is known to change from alpha helix to random coil to beta sheet and vice versa depending on the environment (Arunkumar et al. 1997 Int. J. Biol. Macromol. 21(3):223-230). The conformation may also be influenced by other reagents (Mirtic and Grdadolnik 2013 Biophys. Chem. 175-176 p. 47-53) and potentially by catalysts. These influences can remain dynamic until the reaction terminates.
Some examples of previously synthesized polymers will be described below.
Example 6 of U.S. application Ser. No. 11/613,183 describes polylysine with 22% saturated with methoxypoly(ethylene glycol) (MPEG), by using MPEG succinimidyl-succinate or pre-activated N-hydroxysuccinimidyl polyethylene glycol (NHS-PEG) to saturate polylysine to 22%, which is materially different from the present disclosure (see below) that uses freshly activated MPEG-carboxyl using NHSS (N-hydroxysuccinimidesulfate) and EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) to make solution B, which produces polylysine saturated to 50-60%. Additionally, Example 6 of U.S. application Ser. No. 11/613,183 modified 0.0228 mmol (20 mg) primary amino equivalent, or 0.104 mmol original primary amino based on 22% saturation of said product, by saturating the remaining primary amino with lauric acid after purification. The process of saturation used lauric acid equivalent to 2.4×mol of the original primary amino that was activated with NHSS equivalent to 1.1×mol of the original primary amino and EDC equivalent to 5×mol of the original primary amino. Therefore, Example 6 of U.S. application Ser. No. 11/613,183 is a completely different process compared to the present disclosure (see below) based on the reagents used and their ratios. The product is also a totally different product based on what can be measured analytically such as determination of primary amino groups using trinitrobenzenesulfonic acid (TNBS) giving only 22% PEG saturation.
Example 7 of U.S. application Ser. No. 11/613,183 describes polylysine with 22% saturated with MPEG by using MPEG succinimidyl-succinate or pre-activated NHS-PEG to saturate polylysine to 22% which is materially different from the present disclosure (see below) that uses freshly activated MPEG-carboxyl using NHSS and EDC (solution B) to produce polylysine saturated to 50-60%. Additionally, Example 7 of U.S. application Ser. No. 11/613,183 modified 0.0228 mmol (20 mg) primary amino equivalent, or 0.104 mmol original primary amino based on 22% saturation of said product, by saturating the remaining primary amino with stearic acid after purification. The process of saturation used stearic acid equivalent to 1.71×mol of the original primary amino that was activated with NHSS equivalent to 1.1×mol of the original primary amino and EDC equivalent to 5×mol of the original primary amino. Therefore, Example 7 of U.S. application Ser. No. 11/613,183 is a completely different process compared to the present disclosure (see below) based on the reagents used and their ratios. The product is also a totally different product based on what can be measured analytically such as TNBS giving only 22% PEG saturation.
Example 8 of U.S. application Ser. No. 11/613,183 describes polylysine with 22% saturated with MPEG by using MPEG succinimidyl-succinate or pre-activated NHS-PEG to saturate polylysine to 22%, which is materially different from the present disclosure (see below) that uses freshly activated MPEG-carboxyl using NHSS and EDC (solution B) to produce polylysine saturated to 50-60%. Additionally, Example 8 of U.S. application Ser. No. 11/613,183 modified 0.0228 mmol (20 mg) primary amino equivalent, or 0.104 mmol original primary amino based on 22% saturation of said product, by saturating the remaining primary amino with caprylic acid after purification. The process of saturation used caprylic acid equivalent to 3.36×mol of the original primary amino that was activated with NHSS equivalent to 1.1×mol of the original primary amino and EDC equivalent to 5×mol of the original primary amino. Therefore, Example 8 of U.S. application Ser. No. 11/613,183 is a completely different process compared to the present disclosure (see below) based on the reagents used and their ratios. The product is also a totally different product based on what can be measured analytically such as TNBS giving only 22% PEG saturation.
Example 9 of U.S. application Ser. No. 11/613,183 describes polylysine with 55% saturated with MPEG by using MPEG succinimidyl-succinate or pre-activated NHS-PEG to saturate polylysine to 55%, which is materially different from the present disclosure (see below) that uses freshly activated MPEG-carboxyl using NHSS and EDC (solution B) to produce polylysine saturated to 50-60%. Additionally, Example 9 of U.S. application Ser. No. 11/613,183 modified 0.0318 mmol (40 mg) primary amino equivalent of polylysine-polyethylene glycol (PLPEG), or 0.450 mmol original primary amino based on 55% saturation of said PLPEG product, by saturating the remaining primary amino of said PLPEG product with lauric acid after purification. The process of saturation used lauric acid equivalent to 1.8×mol of the original primary amino that was activated with NHSS equivalent to 0.34×mol of the original primary amino and EDC equivalent to 1.16×mol of the original primary amino. Therefore, Example 9 of U.S. application Ser. No. 11/613,183 is a completely different process compared to the present disclosure (see below) based on the reagents used and their ratios. The product is also a totally different product based on the presence of lauric acid or C12.
Example 12 of U.S. application Ser. No. 11/613,183 used 1 g of Polylysine to make solution A. MPEG-succinate (5 g, 0.59×mol equivalent of the original primary amino) was activated for 18-20 min with 250 mg NHSS (1.15 mmol or 0.68×mol equivalent of the original primary amino) and EDC (2.6 mmol or 1.53×mol equivalent of the original primary amino) to make solution B. Solution C is made by mixing solutions A and B. After 4 hours a second solution B was prepared and added to solution C and the reaction was allowed to incubate overnight. This results in the saturation of epsilon primary amino group of polylysine to 55%. The final amounts of MPEG-succinate, NHSS, and EDC contained in solution C are 1.18×mol, 1.36×mol, and 3.06×mol equivalent of the original primary amino respectively. Compared to the present disclosure (see below) these steps of the process have different ratios and timing of reagent addition. The PLPEG product was purified, lyophilized, and the remaining primary amino groups were saturated with stearic acid by dissolving the purified PLPEG in 143 mL dichloromethane with 2 mmol triethylamine (0.76×mol equivalent of the original primary amino) and adding 2 mmol (0.76×mol equivalent of the original primary amino) of a freshly activated crude C18-NHS in dimethylformamide. The resulting product was purified and tested for GLP-1 binding and was found to have 33% free at 10% loading (see FIG. 37 of U.S. application Ser. No. 11/613,183). When the product of the present disclosure was loaded with GLP-1 at 10% loading no free GLP-1 was observed (see Table 61 below) indicating that the process outlined in Example 12 of U.S. application Ser. No. 11/613,183 produced a product that is different from the present disclosure.
In Example 13 of U.S. application Ser. No. 11/613,183, the purified PLPEG (3 g) with 55% saturation of the primary amino groups used in Example 12 of U.S. application Ser. No. 11/613,183 was saturated with lignoceric acid using a process similar to Example 12 of U.S. application Ser. No. 11/613,183. Again, the process and the product of this process are different from the present disclosure based on the presence of lignoceric acid.
Examples 1-3 of U.S. application Ser. No. 11/971,482 outline processes that use pre-activated NHS-PEG thus these are different processes than the present disclosure. In addition, Examples 1 and 3 have 27% and 22% saturation, respectively, and therefore the product is different from the process of the present disclosure. Example 2 has 55% saturation but is produced using NHS instead of NHSS, as in the present disclosure, so it is a different process and the product may have different PEG distribution along the polylysine backbone.
Examples 4-5 of U.S. application Ser. No. 11/971,482 used 1 g of polylysine with 2.4 mmol primary amine to make solution A in 200 mM HEPES. MPEG-carboxyl (5 g, 0.42×mol equivalent of the original primary amino) was activated for 20 min with 250 mg NHSS (1.15 mmol or 0.48×mol equivalent of the original primary amino) and 500 mg EDC (2.6 mmol or 1.1×mol equivalent of the original primary amino) to make solution B. Activated solution B was added to solution A to make solution C. After 2 hours, a second solution B was prepared and added to solution C and the reaction was allowed to incubate overnight. This results in the saturation of epsilon primary amino groups of polylysine to 56%. The final amounts of MPEG-carboxyl, NHSS, and EDC contained in solution C are 0.84×mol, 0.96×mol, and 2.2×mol equivalent of the original primary amino respectively. Compared to the present disclosure (see below) these steps of the process have different solution C final ratios, in addition to different timing of reagent addition. The PLPEG product with 56% saturation was purified and portions were saturated with behenic acid and stearic acid as described below.
Example 5 of U.S. application Ser. No. 11/971,482 describes processes for behenic acid or C22 saturation, where PLPEG was made using a process different from the present disclosure (see below); PLPEG equivalent to 1.1 of original primary amine was dissolved in 53 mL dichloromethane with 200 μL or 1.44 mmol triethylamine (1.3×mol equivalent of the original primary amino) and 2.5 mmol (2.3×mol equivalent of the original primary amino) of freshly activated crude C22-NHS in 30 mL dimethylformamide:dichloromethane. This addition of C22 was repeated for a second time and allowed to react overnight and the product purified. This process is different from the present disclosure (see below) based on reagents used and their proportions, and results in a product with behenic acid which is different from the product of the present disclosure (see below).
Example 5 of U.S. application Ser. No. 11/971,482 describe processes for stearic acid or C18 saturation, PLPEG was made using a process different from the present disclosure (see below); PLPEG equivalent to 1.1 of original primary amine was dissolved in 53 mL dichloromethane with 200 μL or 1.44 mmol triethylamine (1.3×mol equivalent of the original primary amino) and 2.5 mmol (2.3×mol equivalent of the original primary amino) of freshly activated crude C18-NHS in 30 mL dimethylformamide:dichloromethane. This addition of C18 was repeated for a second time and allowed to react overnight and the product purified. This process is different from the present disclosure based on reagents used and their proportions. Functionally, when loaded with GLP-1 at 2% the product of this process gives 5% free peptide (see Table 1 of U.S. application Ser. No. 11/971,482 and Castillo et al. Pharm. Res. 2012 Vol 29(1) p 306-318) whereas the present disclosure at 2% loading gives 0% free peptide; in fact even at 5 and 10% loading the product of the present disclosure still shows 0% free indicating that the product of the present disclosure has a very high capacity for GLP-1 binding (see below). One is certain that the difference in properties or binding potency can only be explained by differences in composition.
Example 6 of U.S. application Ser. No. 11/971,482 used 1 g of polylysine with 2.4 mmol primary amine to make solution A in 200 mM HEPES. MPEG-succinate (5 g, 0.42×mol equivalent of the original primary amino) was activated for 20 min with 250 mg NHSS (1.15 mmol or 0.48×mol equivalent of the original primary amino) and 500 mg EDC (2.6 mmol or 1.1×mol equivalent of the original primary amino) to make solution B. Activated solution B was added to solution A to make solution C. After 2 hours a second solution B was prepared and added to solution C and the reaction was allowed to incubate overnight. This results in the saturation of epsilon primary amino group of polylysine to 57% with a hydrodynamic diameter of 14 nm. The final amounts of MPEG-carboxyl, NHSS, and EDC contained in solution C are 0.84×mol, 0.96×mol, and 2.2×mol equivalent of the original primary amino respectively. Compared to the present disclosure (see below) this process has different final proportions in solution C as well as different timing of reagent addition and thus the exact organization of PEG on the PL backbone must be different based on the properties of the final product after stearic acid saturation. The PLPEG product with 57% saturation was lyophilized and extracted four times with 50 mL dichloromethane and saturated with 2×2.5 mmol (2×mol equivalent of the original primary amino) C18-NHS dissolved in 30 mL of 1:2 vol/vol of dimethylformamide:dichloromethane after the addition of 400 μL or 2.88 mmol triethylamine (2.6×mol equivalent of the original primary amino). This product was made using a process that is different from the present disclosure (see below) and produces a product that has different potency (binding to GLP-1 at 2% loading has 5% free, see table 1 of U.S. application Ser. No. 11/971,482) compared to the product of the present disclosure (binding to GLP-1 at 2%, 5%, and 10% loading has 0% free). One is certain that the difference in properties can only be explained by differences in composition of the product.
Castillo et al. Pharm. Res. 2012 Vol 29(1) p 306-318 used 1 g polylysine with 2.6 mmol primary amino dissolved in 25 ml of 1 M HEPES, pH 7.4 to make solution A. Methoxy polyethylene glycol carboxymethyl (2 mmol or 0.77×mol equivalent of the original primary amino) was dissolved in 25 ml of 10 mM MES pH=4.7 with 4 mmol NHSS (1.54×mol equivalent of the original primary amino), and, once dissolved, EDC (6 mmol or 2.3×mol equivalent of the original primary amino) was added while stirring to make solution B. Activation was allowed to proceed for 20 min, and the activated MPEG-CM was added directly to the 20PL solution to make solution C. The pH of the solution was adjusted to 7.7 using NaOH and stirred for 2 h at room temperature. An aliquot was taken, and primary amino groups were measured by TNBS and found at 54% MPEG-CM saturation with hydrodynamic diameter of 14.4 nm. The crude PLPEG product was lyophilized and dissolved in ˜100 ml dichloromethane and insoluble precipitates were removed and further extracted with ˜50 ml dichloromethane. The supernatants were pooled, C18-NHS (1.4×mol equivalent of the original primary amino) in 20 mL dichloromethane was added to the pooled supernatant with magnetic stirring, then N,N-diisopropylethylamine (DIPEA, 2.3×mol equivalent of the original primary amino) was added and allowed to react for 4 h. Additional C18-NHS (3.6 mmol or 1.4×mol equivalent of the original primary amino; with total C18-NHS added of 2.8×mol equivalent of the original primary amino) was added and allowed to react overnight to obtain a crude co-polymer product which was purified by ultrafiltration after solvent change to ethanol-water. This process described by Castillo et al. in Pharm. Res. 2012 Vol 29(1) p 306-318 is different and has completely different ratios of reagents compared to the present disclosure. In addition, the resulting purified co-polymer product has different binding properties or potency (binding to GLP-1 at 2% loading has 5% free) compared to the product of the present disclosure (binding to GLP-1 at 2%, 5%, and 10% loading has 0% free; see Table 59) indicating a unique product composition.